En460

The human breast cancer lines MDA-MB-157, MDA-MB-231, MDA-MB-468 and MCF7, the human pancreas cancer cell line MIAPaCa2 and the human gastric cancer cell line MKN45 were purchased from ATCC (Manassas, VA, USA). MDA-MB-157 cells were cultured in Roswell Park Memorial Institute-1640 (Sigma-Aldrich, St Louis, MO, USA) and MDA-MB-231, MDA-MB-468, MCF7, MIAPaCa2 and MKN45 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) at 37 °C in 5% CO₂. Short-hairpin RNA for human ERO1-α (TR313168) was purchased from OriGene (Rockville, MD, USA) and transfected to MDA-MB-231, MCF7 and MIAPaCa2 cells using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). To establish cells with ERO1-α-overexpression, MDA-MB-231 cells were transfected with human ERO1-α cDNA using Lipofectamine 2000 (Life Technologies) as per the manufacturer's instructions. Cells were stably propagated under puromycin selection (1 or 2 μg ml^−l). The ERO1-α inhibitor EN460 was purchased from Millipore (Billerica, MA, USA).